Tuberculosis (TB) has remained a global health problem despite the availability and widespread use of BCG as a TB vaccine, and the existence of effective sterilizing chemotherapy for drug sensitive forms of the disease. In fact, the TB problem has worsened in recent years in many areas of the world. The global HIV epidemic has continued to grow, and it has disproportionately affected resource-limited countries in Africa and Asia. In addition to increased incidence of infection, MDR (Multi Drug Resistant)-TB and XDR (Extensively drug Resistant)-TB have emerged as forms of TB that are significantly more difficult, if not impossible, to treat. Clearly, novel vaccines and novel chemotherapies are urgently needed. The development of these therapies requires new knowledge about the biology of M. tuberculosis infection. By understanding how M. tuberculosis evades immune effectors and drugs, we propose to develop such therapies. Genetics is central to all four Projects. Project 1 (W. Jacobs, PI) seeks to engineer attenuated M. tuberculosis and M. smegmatis mutants as vaccine candidates that have enhanced immunogenicity by incorporating mutations that are required to evade adaptive and innate immunity. Project 2 (S. Porcelli, PI) studies the T cell responses that are required for efficacious vaccines and develops novel screens to find how M. tuberculosis evades T cell responses. Project 3 (J. Chan, PI) examines the role that B cells play in M. tuberculosis immunity and the role of TNF-alpha in acute and long term immunity. Project 4 (J. Blanchard, Pl) will explore the enzymological basis for resistance to penicillins, and refine a novel therapy that employs meropenums and beta-lactamase inhibitors that is potentially highly effective against XDR-TB. All four projects synergize and cross fertilize one another to understand how M. tuberculosis evades killing by drugs or immune effectors with the aims of developing novel therapies to treat TB and XDR-TB. PROJECT 1: Title: Genetic Approaches for Improving Tuberculosis Vaccines Project Leader: Jacobs, W. PROJECT 1 DESCRIPTION (provided by applicant): An efficacious and safe vaccine is needed for Tuberculosis. The goal of this project is to develop novel TB vaccine candidate strains of M. tuberculosis and M. smegmatis. Safety is achieved by generating attenuated mutants that fail to grow in immunocompromised mice. To generate a more efficacious TB vaccine, this project will generate mutants that have enhanced immunogenicity. Enhanced immunogenicity can be achieved by identifying mutations that inactivate determinants that evade host innate and adaptive immune responses. M. tuberculosis has the ability to actively suppress the induction of TH1 cytokines. Aim 1 utilizes a screen employing a macrophage cell line containing the gene encoding the green fluorescent protein fused to the IL-12 inducible promoter. Transposon and null deletion mutants of attenuated M. tuberculosis will be screened for the ability to induce IL-12. M. tuberculosis actively prevents apoptosis. Aim 2 uses a genetic screen to identify apoptotic blocking effectors (abe) mutations. The mutants isolated in both Aim 1 and Aim 2 will be evaluated for their efficacy in protecting mice against aerosolized M. tuberculosis challenges using both M. tuberculosis H37Rv and the KZN strain from South Africa. Preliminary data for Aim 3 has identified a novel set of genes in M. smegmatis named ike for innate killing evasion. The M. smegmatis ?ike deletion Mice immunized with M. smegmatis ?ike mutant containing a set of M. tuberculosis genes (named IKEPLUS) elicits a bactericidal immunity against M. tuberculosis. Heterologous prime and boosts with ILEPLUS and attenuated M. tuberculosis will be explored.